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J. Biol. Chem., Vol. 280, Issue 32, 28877-28884, August 12, 2005

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Plant -Glutamyl Hydrolases and Folate Polyglutamates

CHARACTERIZATION, COMPARTMENTATION, AND CO-OCCURRENCE IN VACUOLES^*

Giuseppe Orsomando, Rocío Díaz de la Garza, Brian J. Green, Mingsheng Peng, Philip A. Rea, Thomas J. Ryan¶, Jesse F. Gregory, III||, and Andrew D. Hanson**

From the Horticultural Sciences and ^||Food Science and Human Nutrition Departments, University of Florida, Gainesville, Florida 32611, Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and ^¶Wadsworth Center, New York State Department of Health, Albany, New York 12201

-Glutamyl hydrolase (GGH, EC 3.4.19.9) catalyzes removal of the polyglutamyl tail from folyl and p-aminobenzoyl polyglutamates. Plants typically have one or a few GGH genes; Arabidopsis has three, tandemly arranged on chromosome 1, which encode proteins with predicted secretory pathway signal peptides. Two representative Arabidopsis GGH proteins, AtGGH1 and AtGGH2 (the At1g78660 and At1g78680 gene products, respectively) were expressed in truncated form in Escherichia coli and purified. Both enzymes were active as dimers, had low K_m values (0.5–2 µM) for folyl and p-aminobenzoyl pentaglutamates, and acted as endopeptidases. However, despite 80% sequence identity, they differed in that AtGGH1 cleaved pentaglutamates, mainly to di- and triglutamates, whereas AtGGH2 yielded mainly monoglutamates. Analysis of subcellular fractions of pea leaves and red beet roots established that GGH activity is confined to the vacuole and that this activity, if not so sequestered, would deglutamylate all cellular folylpolyglutamates within minutes. Purified pea leaf vacuoles contained an average of 20% of the total cellular folate compared with 50 and 10%, respectively, in mitochondria and chloroplasts. The main vacuolar folate was 5-methyltetrahydrofolate, of which 51% was polyglutamylated. In contrast, the principal mitochondrial and chloroplastic forms were 5-formyl- and 5,10-methenyltetrahydrofolate polyglutamates, respectively. In beet roots, 16–60% of the folate was vacuolar and was again mainly 5-methyltetrahydrofolate, of which 76% was polyglutamylated. These data point to a hitherto unsuspected role for vacuoles in folate storage. Furthermore, the paradoxical co-occurrence of GGH and folylpolyglutamates in vacuoles implies that the polyglutamates are somehow protected from GGH attack.

Received for publication, April 20, 2005 , and in revised form, June 14, 2005.

^* This work was supported in part by the Florida Agricultural Experimental Station, by an endowment from the C. V. Griffin Sr. Foundation, and by National Institutes of Health Grant R01 GM071382-01, National Science Foundation Grant MCB-0443709, and United States Department of Energy (Energy Biosciences) Grant DE-FG02-91ER20055. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. 1 and 2.

^** To whom correspondence should be addressed: Horticultural Sciences Dept., University of Florida, P. O. Box 110690, Gainesville, usbFL 32611. Tel.: 352-392-1928; Fax: 352-392-5653; E-mail: adha{at}mail.ifas.ufl.edu.

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